L-cysteine is an amino acid that plays an important role in sulfur metabolism in all living organisms. It is used in the biosynthesis of proteins, such as hair keratin, glutathione, biotin, methionine, and other sulfur-containing metabolites as well as serving as a precursor of coenzyme A. In addition, the biosynthesis of cysteine is known to be closely associated with the biosynthesis of other amino acids including L-serine, L-glycine, and L-methionine. Industrially, L-cysteine and its derivatives find applications in a variety of fields including the pharmaceutical industry (for treatment of bronchial diseases), the cosmetics industry (in hair shampoo, compositions for permanent waves, etc.), and the food industry (antioxidants, flavorant enhancers, dough aids, etc.).
Traditionally, L-cysteine was obtained industrially by acid hydrolysis of human hair or animal feathers (Biotechnology of the Amino Acids Production edited by Ko Aida, p 217-223, 1986). However, not only does the production of cysteine from hair or feathers ensure a yield of as low as 7-8%, but also the use of hydrochloric acid or sulfuric acid produces a significant amount of environment polluting waste. Further, consumers may have a strong aversion to extraction from hair or feathers. These problems have caused a push for the development of environmentally friendly production processes of L-cysteine, leading to the fermentation of L-cysteine utilizing microorganisms.
Representative among the microbial production of L-cysteine is the biological conversion of D, L-ATC using a microorganism (Ryu O H, Ju J Y, and Shin C S, Process Biochem., 32:201-209, 1997). This conversion process is, however, difficult to apply industrially due to the low solubility of the precursor D, L-ATC. Another method of L-cysteine production is direct fermentation using E. coli (Patent No. EP 0885962B; Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48-54, 2006). Excessive accumulation of L-cysteine within microorganisms incur intracellular toxicity, resulting in the limitation for production of L-cysteine at a high concentration. To overcome this drawback, L-cysteine-exporting proteins were employed, however there have been no significant improvements in productivity.
Referring to the biosynthesis pathway of L-cysteine in bacteria and plants, O-acetyl-serine (OAS) acts as an intermediate precursor providing the carbon backbone of L-cysteine (Kredich N M and Tomkins G M, J. Biol. Chem., 241: 4955-4965, 1966). The enzyme O-acetylserine sulfhydrylase (OASS), using hydrogen sulfide as a sulfur donor, catalyses the conversion of O-acetylserine to S-sulfocysteine and finally to cysteine, releasing acetate. Alternatively, SO4 may be reduced to thiosulfate for use as a sulfur donor in cysteine production (Nakamura T, Kon Y, Iwahashi H, and Eguchi Y, J. Bacteriol., 156: 656-662, 1983). The cysteine biosynthesis pathway via OAS use the two enzymes of serine acetyltransferase (CysE), which catalyzes the conversion of serine to OAS, and cysteine synthase (CysK), which catalyzes the conversion of OAS to cysteine. Notably among them serine acetyltransferase (CysE) is highly sensitive to feedback inhibition by the final product cysteine (Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48-54, 2006). Therefore, an altered enzyme that is insensitive to feedback inhibition is needed, however is difficult to develop.